Hindawi Publishing Corporation
International Journal of Vascular Medicine
Volume 2012, Article ID 605101, 7 pages
Anti-Inflammatory Effectsof EpoxyeicosatrienoicAcids
Scott J. Thomson, AraAskari,andDavidBishop-Bailey
Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
Correspondence should be addressed to Scott J. Thomson, firstname.lastname@example.org
Received 30 May 2012; Accepted 20 June 2012
Academic Editor: Ken-ichi Aihara
Copyright © 2012 Scott J. Thomson et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Epoxyeicosatrienoic acids (EETs) are generated by the activity of both selective and also more general cytochrome p450
(CYP) enzymes on arachidonic acid and inactivated largely by soluble epoxide hydrolase (sEH), which converts them to their
corresponding dihydroxyeicosatrienoic acids (DHETs). EETs have been shown to have a diverse range of effects on the vasculature
including relaxation of vascular tone, cellular proliferation, and angiogenesis as well as the migration of smooth muscle cells. This
paper will highlight the growing evidence that EETs also mediate a number of anti-inflammatory effects in the cardiovascular
system. In particular, numerous studies have demonstrated that potentiation of EET activity using different methods can inhibit
inflammatory gene expression and signalling pathways in endothelial cells and monocytes and in models of cardiovascular
diseases. The mechanisms by which EETs mediate their effects are largely unknown but may include direct binding to peroxisome
proliferator-activated receptors (PPARs), G-protein coupled receptors (GPCRs), or transient receptor potential (TRP) channels,
which initiate anti-inflammatory signalling cascades.
Cardiovascular diseases such as atherosclerosis have a strong
inflammatory component. Inflammation in the vascular
wall may be initiated by endothelial dysfunction and
the accumulation of toxic oxidised circulating lipids .
Inflammatory mediators such as TNFα and IL-1β secreted,
which induces the upregulation of cell adhesion molecules,
facilitates leukocyte recruitment in to the vascular wall [2, 3]
and stimulates vascular smooth muscle cell migration and
proliferation . Circulating monocytes not only respond
to inflammatory stimuli by producing large amounts of
activation of lymphocytes and adaptive immunity. The hall-
are monocyte/macrophage rich and highly inflammatory.
Inflammatory responses are normally promptly termi-
nated since excessive or prolonged inflammation can lead
to chronic pathological conditions such as cardiovascular
diseases, Crohn’s disease, rheumatoid arthritis, or cancer.
Although there have been many new treatments recently
developed to combat inflammatory diseases, some of these
treatments are either very expensive and/or not effective
in subsets of patients. Therefore, it is important to con-
tinue to investigate mechanisms that regulate inflammatory
responses as they may open up novel therapeutic targets.
There is a growing list of evidence that the epoxygenase
pathway of arachidonic acid metabolism, which generates
epoxyeicosatrienoic acids (EETs), exerts anti-inflammatory
effects that may be harnessed to treat disease. This paper
will summarise that evidence and highlight outstanding
questions that remain to be answered.
2.Overview of the Epoxygenase Pathway of
Arachidonic acid is an omega-6 polyunsaturated long chain
fatty acid that contains 20 carbon atoms and four cis-
double bonds and possesses a carboxyl group and a methyl
group at respective ends of the molecule. The double bonds
are located between carbons 5-6, 8-9, 11-12, and 14-15
relative to the carboxyl group. Therefore, its chemical name
is all-cis-5,8,11,14-eicosatetraenoic acid and its lipid name
2International Journal of Vascular Medicine
is 20:4 (n-6). The cis-configuration of the four double
bonds causes the arachidonic acid backbone to significantly
bend. In contrast, double bonds in the trans-configuration
or saturated arachidonic acid result in structurally unbent or
Experiments performed more than 30 years ago showed
that incubations of radio-labelled arachidonic acid with
microsomal preparations derived from a variety of tissues
including liver [5, 6], kidney , hypothalamus , and
anterior pituitary  resulted in the formation of EETs.
This “epoxygenase” reaction required cytochrome p450
(CYP) enzymes and utilised NADPH and oxygen in a 1:1
stoichiometric ratio . One atom of molecular oxygen
is incorporated into one of the four double bonds of
arachidonic acid retaining the cis-geometry and yielding
four potential EETs, that is, 5,6-EET, 8,9-EET, 11,12-EET,
or 14,15-EET, respectively. Furthermore, each EET can be
present in either the S/R or R/S stereoconfiguration, thus
eight potential EETs can be formed.
2.1. Epoxygenation of Arachidonic Acid Performed by Spe-
cific CYPs. CYP enzymes catalyze the oxidation of organic
substances, as well as xenobiotics. Altogether, 57 putative
CYP genes have been identified in man (by comparison
mice have 103 and rat 89 CYP, resp.) that are divided into
15 subfamilies . Attempts have been made to classify
human CYP genes by substrate; however, a more systematic
nomenclature is generally used since the true physiological
roles of many of these genes are still unknown . To
date, at least 12 human CYP genes have been reported to
possess epoxygenase activity, although most studies have
been focussed on the CYP2C and CYP2J families, which are
considered the major epoxygenase enzymes.
2.2. CYP2C. One of the earliest studies using recombinant
human CYP compared the metabolic profiles of the CYP2C8
and CYP2C9 enzymes , which are 77% identical at the
amino acid level. Despite their high similarity, CYP2C8 and
CYP2C9 exhibit both regio- and stereoselective differences
in their epoxygenation of arachidonic acid. For instance,
CYP2C8 produced only 14,15-EET and 11,12-EET at a
1.25:1 ratio, which represented 68% of the total metabolites
measured. By contrast, CYP2C9 produced 14,15-EET 11,12-
EET and 8,9-EET at a ratio of 2.3:1:0.5, which represented
69% of total metabolites. Furthermore, with respect to stere-
oselectivity, CYP2C8 was 81% selective for the 11(R),12(S)-
EET configuration, whereas CYP2C9 was 70% selective for
also carry out other reactions including allylic hydroxylation
on arachidonic acid and other fatty acids.
2.3. CYP2J2. Epoxygenase activity of human CYP2J2 was
first demonstrated by the Zeldin lab, who initially cloned
and characterised the gene . Recombinant CYP2J2
metabolised arachidonic acid to all four potential epoxy-
genase products, with 14,15-EET being the predominant
in the heart, and EETs were produced in similar proportions
as recombinant CYP2J2 suggesting that CYP2J2 played a
major role in EET generation in the heart in vivo .
CYP2J2 expression is also seen in kidney, liver and muscle
tissues , and, to a lesser extent, in the gut .
2.4. Other CYPs. A comprehensive comparison study by
Rifkind and colleagues examined the epoxygenase activity of
a panel of 10 CYP proteins by overexpressing them in HepG2
cells and measuring metabolic products. CYP 2C8, 2C9,
contrast, CYP2D6 was inactive, while CYPs 2A6, 3A3, 3A4,
and 3A5 had minimal epoxygenase activity . CYP3A4
has also been shown to make the epoxygenase products 8,9-
cancer cell lines . Other CYPs that have been shown
to possess epoxygenase activity include CYP1A, CYP2B1
and CYP2B2  and CYP2B12 , CYP2C8, CYP2C9,
CYP2D18 , CYP2N1 and CYP2N2 , and rat CYP4A2
and 4A3 . The full extent of the epoxygenase activity
of these enzymes and the physiological consequences of any
activity is, however, poorly understood.
Once formed, EETs are unstable because they are rapidly
EETs into dihydroxyeicosatrienoic acid (DHETs), catalysed
by soluble epoxide hydrolase (sEH) . DHETs are gen-
erally considered to be less active; however, they have been
shown to exert vasodilatory effects on coronary arteries .
DHETS are far more polar than their corresponding EETs
and quickly diffuse out of tissues as the 1, diols or conjugates
of them. Other pathways of EET metabolism include chain
elongation, β-oxidation, and ω-oxidation . 5,6 EET and
8,9 EET are substrates for COX enzymes  and can also be
incorporated into membrane phospholipids. DHETs have a
lower binding affinity for phospholipids which may account
for its relatively increased plasma levels .
Recently, the damaging cardiovascular risk factor homo-
cysteine has been shown to upregulate sEH in endothelial
cells and promote a proinflammatory environment . In
contrast, elevating the levels of endogenous CYP products by
hydrolase (sEH-1) has been shown to reduce neointima for-
mation , atherosclerosis and abdominal aortic aneurysm
development, dyslipidaemia in hyperlipidaemic mice ,
and reduce hypertension  and diabetes  in different
mouse models. A number of sEH inhibitors have now been
developed and are moving towards clinical trials for a variety
of disorders related to cardiovascular disease.
CYP2C mediated generation of 11,12 EET has also been
documented in porcine coronary arteries , and CYP2C
enzymes have been found expressed in endothelial cells ,
International Journal of Vascular Medicine3
and in primary human monocytes and M1 (CYP2C8) and
M2 macrophages (CYP2C8 and CYP2C9) .
CYP2J2 immunoreactivity is seen in the endothelial and
well as in the human monocytic cell lines THP-1 and U937,
primary monocytes and M1 and M2 macrophages ,
and the endothelial cell line EA hy.926s . Interestingly,
neither CYP2J2 nor CYP2C8 mRNA expression was detected
in human polymorphonuclear cells . More recently, the
increased risk of coronary artery disease was shown to be
associated with a polymorphism in the promoter of CYP2J2
gene in some populations, which decreases the expression of
the enzyme .
EETs have been shown to exert multiple biological effects
on the vasculature including proproliferation and angio-
genic effects . EETs have also been hypothesized as
endothelium-derived hyperpolarizing factors, as they hyper-
polarize and relax vascular smooth muscle cells by activat-
ing calcium-activated potassium channels . However,
a number of the anti-inflammatory activities of EETs on
inflammatory cells, as discussed below, appear independent
of any cellular hyperpolarisation .
5.1. Endothelial Cells. Overexpression of CYP2J2 in human
and bovine endothelialcells
VCAM-1  and VCAM-1 promoter activity in reporter
assays . Treatment with the epoxygenase inhibitor
SKF525A reversed the effects of CYP2J2 overexpression on
VCAM-1 promoter activity . Exogenous EETs also exert
EETs can have different selectivities. In human endothelial
cells, 11,12-EET significantly inhibited VCAM-1 expression
in response to TNFα, IL-1α, or LPS. By contrast, 14,15-EET
had negligible effect, while 5,6-EET, 8,9-EET, and 11,12-
DHET all inhibited to varying degrees but to a lesser extent
than 11,12-EET. 11,12-EET also inhibited TNFα-induced
E-selectin and ICAM-1 expression . Mice engineered
to overexpress the human epoxygenase genes CYP2J2 or
CYP2C8, respectively, were generated to investigate their
roles in endothelial cells. Primary pulmonary endothelial
cells derived from these mice showed reduced levels of LPS-
induced adhesion molecule and chemokine gene expression.
Furthermore, these anti-inflammatory effects were inhibited
by treatment with the epoxygenase inhibitor MS-PPOH and
a putative EET receptor antagonist 14,15-EEZE .
has also been shown to antagonise inflammatory signals in
monocytic cells. Phorbol ester treatment of THP-1 led to
a 4-fold increase in CYP2J2 expression between 3–7 days
after stimulation, suggesting that endogenous expression
of CYP2J2 may regulate inflammatory responses in these
cells . Addition of 8,9-EET or 11,12-EET inhibited
basal TNF secretion from THP1 cells by about 90% and
40%, respectively . Similarly, the epoxygenase inhibitor
SKF525A led to a concentration-dependent superinduction
of LPS-induced PGE2in rat monocytes and COX-2 in mouse
and human monocytes . Consistent with these find-
ings, exogenous 11,12-EET dose dependently inhibited LPS-
induced PGE2 and attenuated SKF-mediated superinduc-
tion. 11,12-EET also inhibited LPS-induced COX-2 activity
and expression . EETs can, therefore, both compete with
arachidonic acid for the binding site in COX enzymes as
well as inhibit the inflammation induced induction of COX-
2 expression. A study found that EETs were detected in
human peritoneal macrophages under basal conditions, but
not following zymosan treatment, which caused a shift to
prostaglandin synthesis .
5.3. Leukocyte Endothelial Cell Interactions. Several studies
have demonstrated that EETs can regulate functional inter-
action between leukocytes and endothelial cells. Treatment
of endothelial cells with 14,15-EET significantly enhanced
attachment of the monocytic cell line U937 . Pretreat-
ment of endothelial cells with EETs alone or in combi-
nation with PMA had negligible effects on adherence of
PMNs. However, cotreatment of EETs and PMA led to a
concentration-dependent decrease in adherence of PMNs
when cocultured with endothelial cells . 11,12-EET, but
cells in an ex vivo model. Mice were treated with TNFα
and carotid arteries were removed and incubated with U937
to that of treatment with a blocking VCAM-1 antibody
. PBMCs derived from mice systemically overexpressing
human CYP2J2 via in vivo gene delivery were significantly
5.4. In Vivo Models. There have been conflicting reports
on the effects of EETs in acute models of inflammation in
vivo. Rats injected with TNFα showed elevated plasma levels
of adhesion molecules and inflammatory cytokines, and
decreased levels of the anti-inflammatory mediator IL-10.
However, these effects were significantly reduced by systemic
overexpression of human CYP2J2 , suggesting that EETs
act as anti-inflammatory mediators. Similarly, TNFα-treated
human bronchi also showed reduced inflammation when
treated with 14,15-EET . LPS responses of wild-type
mice have also been compared to sEH−/− null mice or mice
that had endothelial-specific overexpression of the human
CYP2J2 or CYP2C8. All three genetically modified mice
had reduced levels of inflammatory gene expression and
neutrophil recruitment in the lung following LPS injection.
Moreover, these effects correlated with decreased activation
of the key transcription factor NF-κB . By contrast,
another study found that to sEH−/− null mice were not
protected from LPS-induced inflammatory gene expression
or neutrophil recruitment in the liver, and that treatment
with the sEH inhibitor AUDA also had minimal effect liver
4International Journal of Vascular Medicine
or that liver is more susceptible to endotoxin shock.
6.1. NF-κB Inhibition. The mechanisms by which EETs
mediate their anti-inflammatory effects remain ill-defined,
but there are several reports that they can inhibit activation
of NF-κB, a key transcription factor for inflammatory gene
induction. In mammals NF-κB comprises five subunits, with
the RelA (p65) subunit being expressed in most cell types.
Under basal conditions, NF-κB dimers are localised in the
cytoplasm due to interactions with IκB (inhibitor of NF-
κB) proteins. Signalling cascades induced by inflammatory
descend on the IKK (inhibitor of NF-κB kinase) complex,
which phosphorylates IκB. This tags IκB for subsequent
turn, facilitates NF-κB nuclear translocation where it binds
to its cognate binding elements to activate transcription
11,12-EET inhibits NF-κB reporter activity in both
HEK293 cells  and human endothelial cells  fol-
lowing stimulation. Furthermore, 11,12-EET also inhibited
TNFα-induced RelA nuclear translocation, IκBα degrada-
tion, and IKKα activity, respectively , indicating that
EET-mediated inhibition of NF-κB occurs upstream of IKK.
Interestingly, 14,15-EET was also shown to inhibit the
TNFα-induced degradation of IκBα in primary human lung
tissue  but had no effect on NF-κB reporter activity
in HEK293s, suggesting that 14,15-EET may act in a cell
type-specific manner. Similarly, 8,9-EET and 11,12-EET
inhibited NF-κB reporter gene activity in HEK293 cells .
In contrast to CYP2J2, CYP2C9 increased NF-κB activity
in human vascular endothelium via superoxide generation,
potentially giving this CYP a proinflammatory profile .
6.2. STAT3. EETs can also activate STAT3 in human breast
cancer cell lines, with 14,15-EET promoting STAT3 tyrosine-
705 phosphorylation and nuclear translocation . Acti-
vation of STAT3 was shown to be dependent on cell
proliferation, which led the authors to conclude that 14,15-
EET may be involved in an autocrine/paracrine pathway
driving cell growth. Interestingly, the anti-inflammatory
effects of IL-10 in macrophages are also dependent on STAT3
tyrosine-705 phosphorylation . Taken together, these
results suggest that the anti-inflammatory effects of EETs
may be mediated by activation of STAT3, in addition to the
inhibition of NF-κB.
6.3. EETs as PPAR Agonists. PPARs are a subfamily of the
nuclear receptor superfamily that comprises three ligand-
activated transcription factors: PPARα (NR1C1), PPARβ/δ
(NR1C2), and PPARγ (NR1C3). Upon ligand binding, they
form heterodimers with the retinoid X receptor and bind to
specific response elements in gene promoters to upregulate
gene transcription . PPARs have been shown to regulate
diverse physiological processes such as fatty acid and glucose
metabolism, angiogenesis, and cellular proliferation and
differentiation, in addition to inflammation. PPAR ligands
include a variety of fatty acids, and there has been recent
evidence that metabolites of the epoxygenase pathway can
activate PPAR receptors.
The omega-alcohol of 14,15-EET, 20,14,15-HEET, or a
1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs
activated human and mouse PPARα in transient transfection
assays, suggesting a role for them as endogenous ligands
for these orphan nuclear receptors . Overexpression of
human CYP2J2 in HEK293 cells resulted in a synergistic
activation of PPARα, -β/δ and, -γ reporter gene activity.
8,9-EET and 11,12-EET, but not 14,15-EET, (in contrast to
its hydroxy metabolite 20,14,15-HEET) were able to induce
PPARα reporter activity . Furthermore, IL-1β-induced
NF-κB reporter activity and COX-2 mRNA induction in
HEK293 cells was significantly inhibited cells expressing of
CYP2J2 and PPARα.
Competition and direct binding assays subsequently
revealed that EETs bind to the ligand-binding domain of
PPARγ with K(d) in the μM range. In the presence of the
sEH inhibitor AUDA, EETs increased PPARγ transcription
activity in endothelial cells and 3T3-L1 preadipocytes. In
endothelial cells, AUDA enhanced, but overexpression of
sEH reduced laminar flow-induced PPARγ activity, EET
generation, and the inhibition of VCAM-1 expression .
PPARs, therefore, represent a viable receptor target for the
anti-inflammatory effects of EETs. However, it should be
noted that AUDA may exert multiple effects in addition to
sEH inhibition. It has been shown to act both as a PPAR
agonist  and a EET mimetic ; therefore, results using
AUDA should be cautiously interpreted.
6.4. GPCRs. For some time it has been suggested that EETs
might mediate many of their effects via binding to a putative
EET produced a 0.5- to 10-fold increase in the activity of the
KCa channels in smooth muscle cells derived from bovine
coronary arteries, which was dependent on the presence of
GTP . Furthermore, blocking antibodies against GSα,
but not Gβγ or anti-Giα, were able to inhibit the activation
induced by 11,12-EET . Using radio-ligand binding,
14,15-EET has been shown to have a high affinity for a
receptor expressed on guinea pig-derived mononuclear cells,
which was purported to be a G-protein coupled receptor
that stimulated cAMP production . This putative GPCR-
cAMP pathway remains elusive but may represent a novel
anti-inflammatory pathway by which EETs act.
6.5. TRPV1 and EETs. TRPV4 is a cation channel of the
“transient receptor potential” (TRP) family that functions
as a Ca2+entry channel, that is expressed in smooth muscle
cells, endothelial cells, as well as in perivascular nerves.
CYP-dependent generation of 5,6-EET can activate TRPV4
in murine endothelial cells and is a possible contributing
mechanism to the hyperpolarising effects of EETs .
Additionally, 11,12-EET can activate TRPV4 channels in
smooth muscle cells from rat cerebral arteries , and 5,6-
EET and 8,9-EET can activate TRPV4 in human endothelial
International Journal of Vascular Medicine5
cells . Although activated by EETs, there is little evidence
that activation of TRPV4 is anti-inflammatory, though it
does lead to vasodilation via nitric oxide, prostacyclin,
and intermediate/small conductance K+ channel-dependent
pathways, and in vascular smooth muscle, large conductance
K+ channel activation, and hyperpolarization .
More than 100 metabolites derived from arachidonic acid
have been described, with the best characterised com-
ing from the COX and LOX pathways which generate
prostanoids and leukotrienes, respectively . Knowledge
of these pathways has led to several important therapeutic
breakthroughs such as COX inhibitors which are used to
treat pain and inflammation and leukotriene antagonists
that have been used to treat asthma. By contrast, much less
is known about the epoxygenase pathway of arachidonic
acid metabolism, although as outlined in this paper, EETs
can exert a number of cardio-protective anti-inflammatory
effects on vascular cells such as endothelial cells and
monocytes. These include inhibition of proinflammatory
mediators and cell adhesion molecules. Indeed, a recent
study has measured epoxygenase products in atherosclerotic
patients . Compared to healthy volunteers, both obese
and nonobese CAD patients had significantly higher plasma
EETs , suggesting that this is a compensation mechanism
to protect against ongoing vascular inflammation.
Although elevating epoxygenase products via sEH inhi-
bition have been shown to be beneficial in a wide variety
of animal models of cardiovascular disease, the mecha-
nisms through which these effects are mediated are still
largely unknown, although NF-κB and STAT3 have both
been implicated. However, several fundamental question
regarding the role of EETs in vascular inflammation remain
unanswered. Firstly, it is clear that CYP epoxygenases can act
on substrates other than arachidonic acid, such as cardio-
protective fish oils. Eicosapentaenoic acid for example is an
omega-3 long chain fatty acid that differs from arachidonic
acid by the addition of one extra double bond at the 17-
18 carbon position. Epoxygenation of eicosapentaenoic acid
by CYP enzymes generates 17,18-epoxyeicosatrienoic acid,
which has a hyperpolarising effect on bronchial smooth
muscle cells in vitro and in vivo . Similarly, linoleic
acid, which is the major dietary fat, can be epoxygenated
by CYP enzymes resulting in potent metabolites which
are probably proinflammatory in nature. However, little is
known regarding the function of many of these alternative
during inflammation. Secondly, the full range of epoxy-
genase activity by CYP enzymes in healthy and diseased
physiological settings is still not completely understood and
remains a significant barrier to progress in the field. Thirdly,
and probably most importantly, definitive identification of
a specific receptor that mediates the activities of EETs
is essential to fully understand the epoxygenase pathway,
and will help to elucidate new therapies for cardiovascular
diseases in the future.
This work was supported by the British Heart Foundation
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